I have begun my experiment. To tell the truth, it is not a glamorous affair. It wasn’t even my idea, though I wish it was. It consists of nothing more than 5 gallon plastic jugs, black plastic, duct tape, and several HOBO U26 dissolved oxygen data loggers. The idea came out of a planning meeting where the desire to get more precise measurements of microbial production in the rivers flowing into Tillamook Bay was one of many topics discussed. The method proposed was to incubate samples of water in the dark for a period of time, and measure the change in dissolved oxygen from start to finish. Any decrease in the oxygen level should be the result of organisms in the water respiring, eating or decomposing organic material in the water.
It is important to know how bacteria change the conditions of the water, changing the amount of nutrients that flow into the bay and the concentrations of both oxygen and carbon dioxide. The ocean is warming and becoming more acidic, bringing increasingly acidic water into the bay with every incoming tide. The nutrients that flow into the bay from the surrounding watersheds lead to increased bacterial growth that close the oyster beds in the bay to harvesting, as well as further acidifying the waters, as dissolved carbon dioxide increases in the water as bacteria release it as a waste product of their metabolic processes and it reacts with water to form carbonate, bicarbonate or carbonic acid. The relationship between higher nutrient inputs into marine waters from terrestrial systems and increased acidification is not just a bay or estuary problem, but a coastal problem as well. The EPA, along with the Navy and many other players, hope to assemble the big picture of how Tillamook Bay works with respect to all of these issues so that the people of the region can better plan for climate change and mitigate potential problems, as well as improve the health of the ecosystem and the lives that depend on it.
There was a considerable debate about how best to go about incubating the water and how to measure oxygen changes in a scientifically valid manner. The issue with the incubation lies in the transporting of the water to the lab. Changes to the temperature of the water will effect the solubility of oxygen in the water, as well as the rate of production of bacteria, affecting the results. Maintaining water samples at a constant temperature remains impractical and a concern, but a greater concern is with the Winkler method. The Winkler method is a method of analysis that allows for measuring the dissolved oxygen at a single point in time, but not continuously, but the measurement can be more precise than that of a datalogger. It was proposed that only initial and final oxygen measurements would be used to determine the total change in a sample, and the incubation period would be short, a few hours at most. As the experiment was discussed, it became apparent that such a short period of incubation may not adequately capture microbial growth, and that a time series of measurements would be the only valid method of determining a rate of microbial production. Thus, the 5 gallon jug trials, to gather a time series, determine a proper incubation period, and as a basic proof of concept.
Friday we drove to Tillamook and collected samples, though not as many as we had planned to. We are also evaluating an alternate incubation method using 2 gallon ziplock freezer bags kept dark in a cooler. The samples have been incubating all weekend. On Monday I will download the data from the loggers and see what there is to see. I may even have a graph or two to share next time!
Good luck with the alternate trials, Jeremy. What was the final verdict on transportation – all samples in a cooler? Do colder temperatures affect microbial respiration at all?
We plotted the data today and in some ways it was a success; 3 out of 4 showed a pretty linear decrease in dissolved oxygen, but one of the ziplock bags showed a curvilinear decrease and almost flattened. All of the plots show some strange noise in DO, % saturation, pressure, and temperature, and all the weird stuff happened over the weekend while they were sitting in the lab in the dark. We have no solid ideas at this point. Temperature and pressure mediate saturation, and the water samples all experienced a major change in temperature and pressure. The carboys were capped under water at a temperature of 16 celsius, and by the time they warmed to ambient in the lab over the weekend, they were at least 10 degrees warmer. Water expands with increasing temperature, and maybe that change in pressure was enough to monkey with the chemistry, or alter a key parameter that the sensor uses to calculate DO. Microbial respiration is also affected by temperature, but we don’t know if it does so to the degree that it would cause the noise we’re seeing.
One awesome thing we saw on the plot of atmospheric pressure were massive drops in pressure that corresponded with the times we had the sensor in the car with us traveling to and from Tillamook. It was recording the Bernoulli effect on the car at speed! Pretty cool.
Great job explaining the importance of what you’re doing and going through the steps of how to design an experiment, as well as all of the inevitable issues you will run into with that experiment. Your results as outlined in your comment to Sarah show that you will have plenty more brainstorming to do to perfect your methods and explain discrepancies. The sensor recording the Bernoulli effect while in the car is super neat!